The long-term objective of this proposal is to understand how a replisome responsible for DNA replication is assembled from its component proteins and how it functions in the coordination of leading and lagging strand DNA synthesis. The system being examined to gain such insights is the T4 phage replisome derived from eight proteins including a polymerase, clamp-loader complex, clamp, helicase, primase, helicase loader, and single- stranded DNA-binding protein. These proteins can be grouped into subassemblies: the holoenzyme and the primosome reconstituted from the polymerase and clamp proteins, and from the helicase and primase proteins respectively. We are specifically interested in how these subassemblies are formed, their composition and structure, the identity of their protein-protein contacts, their interactions with single-stranded DNA binding protein, their location relative to their DNA templates, their dynamic properties with respect to dissociation from the replisome and their movement at the replication fork. The ultimate objective is to integrate these finding with our proposed observations of a functioning replisome carrying out leading/lagging strand synthesis at the single-molecule level. Answers to these complex questions will be sought with a wide assortment of techniques varying from crystallography to single-molecule and ensemble kinetics. The generality of the findings will be tested by extension of similar experiments to the yeast (Sacchromyces cerevisae) and human holoenzyme. Building on our understanding of holoenzyme assembly and function, the proposed studies will expand to investigate lesion bypass, and how a replisome copes with the problem of a damaged template base in creating a complementary strand. Of particular interest is how a specific lesion bypass polymerase is selected from a pool of Y-class polymerase candidates; the signal for and role of ubiquitination in the switch of a Y-class polymerase for the replicative polymerase; the composition of the bypass holoenzyme complex; and ultimately the pathway for reversal and restoration of the replicative holoenzyme. Investigations will be at the in vitro and cellular level. DNA replication is at the heart of a cell's ability to clonally expand; a deepened understanding of this fundamental process is essential for interpreting the effects of changes in the fidelity and efficiency of replication in a variety of disease states, from viral infection to cancer and for the selection of specific replisomal and bypass proteins as potential therapeutic targets.